1. Field of the Invention
The present invention is related to the fields of molecular biology, developmental biology, physiology, neurobiology and medicine. The invention provides polynucleotides encoding the zebrafish PTH1R receptor and polynucleotides for a novel zebrafish PTH3R receptor, as well as vectors and cells containing said polynucleotides. The invention further provides polypeptides for the PTH1R and PTH3R receptors. The polynucleotides and polypeptides of the invention are useful for the identification of agonist and antagonists of PTH1R or PTH3R receptor function and as reagents in the treatment of diseases or disorders associated with PTH1R or PTH3R function.
2. Related Art
The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) mediates in mammals and frogs the actions of PTH and PTHrP. Both peptides, most likely evolved through a gene duplication event from a common ancestral gene, and have retained limited homology within the amino-terminal region. Because of their structural conservation, PTH and PTHrP bind with similar affinity to the PTH1R, and activate this common receptor with similar or indistinguishable efficacy. Due to this unusual ligand-specificity, the PTH1R mediates the endocrine actions of PTH, the most important peptide regulator of calcium homeostasis in mammals, and the autocrine/paracrine actions of PTHrP, which is important for normal chondrocyte proliferation and differentiation (Karaplis, et al., (1997)); Lanske, et al., (1996)), and most likely for other, still incompletely defined functions including pancreas, skin, and breast development, as well as tooth eruption (Wysolmerski, et al., (1996)).
In addition to the PTH1R, a PTH type-2 receptor (PTH2R) has been isolated from mammals and teleosts (Usdin, et al., (1995); Rubin, et al. (1999)), and at least the human PTH2R is activated by PTH and a recently isolated hypothalamus peptide (Usdin, et al., (1999)). Its biological importance remains uncertain, as does the importance of additional receptors for which there is growing biological and pharmacological evidence. For example, receptors with specificity for amino-terminal PTH and PTHrP have been described for keratinocytes, squamous carcinoma cell lines, and central nervous system cells (Orloff et al., (1995 and 1996); Fukayama, et al., (1995)), and there is evidence for a PTHrP-selective receptor in the mammalian supraoptic nucleus (Yamamoto et al., (1997) and (1998)). In addition, the midregional portion of PTHrP stimulates an increase in intracellular free calcium in some cell lines and increases placental calcium transport (Kovacs et al., (1996); Wu et al., (1996); Orloff et al., (1996)), and the carboxy-terminal portion of PTH binds to a distinct receptor on clonal cell lines (Inomata, et al., (1995); Takasu, et al., (1998)).
Research on the parathyroid hormone (PTH) has been extensive. Formulations of PTH and compounds with PTH activity have been described (see U.S. Pat. Nos. 5,496,801; 5,814,603; 5,208041;), and methods to produce the same are also known (see U.S. Pat. Nos. 5,616,560 and 5,010,010). In addition, analogs and inhibitors of PTH may be found in the prior art (see U.S. Pat. Nos. 4,423,037; 5,693616; 5,695,955 and 5,798,225).
Thus, the invention furthers the art by focusing on PTH receptors and providing reagents and methods that are distinct and separate from PTH.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a novel PTH1R receptor having the amino acid sequence shown in FIG. 2A (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host with the ATCC(copyright) (a trademark of the American Type Culture Collection; hereinafter referred to as ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, as patent deposit PTA-916 on Nov. 4, 1999. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of PTH1R polypeptides or peptides by recombinant techniques. The invention further provides an isolated PTH1R polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding a novel PTH3R receptor having the amino acid sequence shown in FIG. 2B (SEQ ID NO:4) or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host with the ATCC as patent deposit PTA-915 on Nov. 4, 1999. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of PTH3R polypeptides or peptides by recombinant techniques. The invention further provides an isolated PTH3R polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by the PTH1R or PTH3R receptor, which involves contacting cells which express the PTH1R or PTH3R receptor with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on PTH or PTHrP binding to the PTH1R or PTH3R receptor. In particular, the method involves contacting the PTH1R or PTH3R receptor with a PTH or PTHrP polypeptide and a candidate compound and determining whether PTH or PTHrP polypeptide binding to the PTH1R or PTH3R receptor is increased or decreased due to the presence of the candidate compound.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of PTH1R or PTH3R activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated PTH1R or PTH3R polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of PTH1R or PTH3R receptor activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an PTH1R or PTH3R antagonist.
The invention further provides a diagnostic method useful during diagnosis or prognosis of diseases and disorders associated with PTH1R or PTH3R receptor expression or function.